The helix dipole
Jasper Minkels, 0610437
Introduction
Throughout the scientific literature on protein research of the last thirty years, the α-helix dipole is
mentioned in a number of cases where its partial charge on both the C-terminal and the N-terminal
ends is thought to be involved in various biological processes on a molecular level. In order to
understand the cause of this dipole moment, one must look at the structure and the geometry of the
α-helix. The α-helix is a right handed coiled structure. Each amino acid makes a turn of about 100
degrees and hence it requires 3.6 residues to make a full turn. Amino acids that wind around the axis
form hydrogen bounds with each other. The N-H group of each
amino acid forms a hydrogen bond with the C=O of the amino
acid that is located four places earlier in the helix. The vast
number of hydrogen bonds that is formed is actually the
underlying reason for the forming of this secondary protein
structure.
Although the principles behind the occurrence of the
helix dipole have been described earlier, its role in protein
structure and function was first reviewed by Hol in 1985. He
states that the helix dipole originates in the dipole of the
individual peptide unit. The charge distribution within such a
unit is pictured in Figure 1. Its direction is parallel to the N-H
and C=O bonds. It has been shown that in an α-helix around
Figure 1: Peptide charge distribution,
97% of all peptide dipole moments point in the direction of the numbers in units of the elementary
helix axis, the dipole is therefore quite insensitive to the φ and ψ charge[8]
angles. The C=O groups are in a slightly upward direction
(toward the C-terminus) and the N-H groups are in a downward direction (toward the N-terminus)
and this gives rise to a small dipole. The aggregate effect of all individual dipoles in an α-helix is a
negative dipole moment at the C-terminal end, and a positive dipole moment at the N-terminal end
of the helix. The dipole moment of an individual peptide unit is about 3,46 D which equals 0,72 e Å
or 0,5 e per 1,5 Å. Since the axial shift per residue in an α-helix is also 1,5 Å, all dipoles cancel out
except for the C- and N-terminal ones. However, a dipole moment is also affected by the local
environment and especially hydrogen bonds are known to increase the value of dipole moments.
Both calculations and experimental measurements confirm that the dipole moment is indeed
increased, by 25 to 50%.
Although the amount of known protein structures and biological mechanisms has
enormously expanded since then, Hol already distinguished a few roles for the helix dipole in
proteins that still prove to be relevant. In his review he discusses the role of the helix dipole in
glutathione peroxidase, rhodanese, subtilisin, triose phosphate isomerase, glyceraldehyde-3phosphate dehydrogenase, p-hydroxybenzoate hydroxylase, glutathione reductase, thioredoxin and
papain. In these proteins, the helix dipole is used for binding stabilizing (intermediary) charges
varying from anions to active site residues. Hol also analyzed twenty proteins that were known to
bind phosphate groups. The majority of these proteins combined the N-terminal dipole and
positively charged residues for this, although a few proteins such as flavodoxin depended solely on
a helix dipole and neighbouring N-H groups for phosphate binding. It was found that the phosphate
binding helices were often part of a βαβ-motif. However, the individual amino acid sequences
mostly differed, except for a highly conserved glycine that was incorporated to provide space for the
approaching phosphate moiety. Hol thus concluded that the electrostatic interactions, and not a
specific sequence, was essential to the phosphate binding mechanism. Further research aimed to
analyze if the binding by these anions was due to the possibility of forming large amounts of
hydrogen bonds concluded this was not the case, thereby confirming his conclusion. At the time no
proteins were known that applied a similar mechanism to DNA or RNA binding.
Hol further concluded in his review that the α-helix dipole may explain the most frequently
observed folding patterns in proteins. He also included the dipole of parallel β-strands in his model,
but further research proved this dipole to be non-existent. His conclusions in summary:
(i) In all-helical proteins, helices tend to be anti-parallel because of a favourable
interaction between alpha-helix dipoles. This also explains the frequent occurrence of
alpha-alpha-units;
(ii) In αβ-proteins, a favourable interaction occurs between the β -dipoles in the center of
the molecule and the α-helix dipoles arranged in an anti-parallel way around the
parallel beta-strands. Such an interaction would also explain the stability of the
βαβ -unit;
(iii) In all-β proteins the favourable αβ-dipole interactions are absent and β-strands tend
to be anti-parallel as an unfavourable interaction between parallel β-dipoles would
otherwise occur.
Finally, Hol performed calculations on the interaction
between two α-helices in vacuum and concluded that the
most important parameters are the helix length, the distance
between the two helices and the Ω-angle. These parameters
are pictured in Figure 2. Hol recognized four different related
environments: bulk water, bulk protein, the outer protein
surface and the proteins first hydration shell. Due to different
mobilities and dielectric properties Hol rightfully concluded
that exact calculations on interactions in proteins aren’t
straightforward as no dielectric constant can be applied. We
will see whether Hol was right in stating that “the α-helix
dipole plays an important role in modulating the properties of Figure 2: Visualization of the parameters
used by Hol[8]
several enzymes and in defining the mode of coenzyme
[8]
binding by numerous proteins”.
Channels
Ion channels
The electrostatic effects of the helix dipole are often described as relevant in multiple classes of ion
channels. Doyle et al. solved the protein structure of the potassium channel from Streptomyces
lividans at a 3,2 Å resolution. Its amino acid sequence is similar to that of other potassium channels,
both voltage gated and cyclic nucleotide gated, in various
organisms. Molecular cloning and mutagenesis experiments
have reinforced the conclusion that all K+ channels have
essentially the same pore constitution, sharing a critical amino
acid sequence that is vital for distinguishing K+ from Na+
ions. This specificity and the high conductivity of the channel,
up to 108 ions per second, are the main points of interest of the
authors. The following information can be extracted from their
article[4]. The potassium channel is a tetramer consisting of
four identical units with two membrane-spanning regions each.
Of these two, one transmembrane helix faces the central pore
while the other faces the lipid membrane. The inner helices are
tilted with respect to the membrane normal by about 25
Figure 3: Ribbon representation of the
degrees and are slightly kinked. The inner helices of all four tetramer as an integral membrane protein.
[4]
subunits pack against each other as a bundle near the
intracellular aspect of the membrane, giving the appearance of
what the authors call an “inverted teepee”[4]. Figure 3 is a schematic representation of the channel.
Negatively charged acidic amino acids are located near both entryways of the channel. This ensures
a high concentration of cations. From inside the cell the pore begins as a tunnel and then opens into
a wide cavity near the middle of the membrane. Up to this part a passing K+ ion can remain largely
hydrated. Along with these water molecules the helix dipoles of the four pore helices, with their
carboxyl end pointed towards the center of the cavity, help to lower the energy barrier although they
are 8 Å away from the cavity center, a point to which we will return later on.
Figure 4 depicts the cavity and the surrounding helices. Whereas the selectivity filter is lined
with polar amino acids, the pore is lined with hydrophobic
groups. The authors propose that the lack of possible
interactions between these groups and the passing ion
improves the throughput rate. Another improvement follows
from the structure of the selectivity filter. It consists of two
binding spots. While one ion is bound quite tight, the arrival
of the second ion on the nearby binding spot repulses the first
one, thereby pushing it through the selectivity filter. A
molecular spring that ensures the widening of the selectivity
filter accounts for the fact that a dehydrated Na+ ion, which
has a 0,95 Å radius compared to 1.33 Å for K+, cannot be
Figure 4: Both the aqueous cavity and the
helices (four in total) stabilize the ion (green) properly stabilized. Hence the fact that the channel is over
in the K+ channel.[4]
10.000 times more permeant to potassium ions.[4]
Roux and MacKinnon quantified the stabilization energies in a similar type of potassium
channel, the KscA K+ channel, using the Born theory of solvation[2]. The free energy for
transferring an ion from bulk water into a cavity of radius R and dielectric constant ε w embedded in
a medium of low dielectric constant ε m is given by:
ΔG = (1 / 2) (Q2 / R) (1 / ε m -1 / ε w)[19]
The transfer of a potassium ion from an aqueous solution to a water filled sphere (radius 5 Å, ε w =
80) surrounded by hydrocarbons (ε m = 2, which corresponds to a 25 Å membrane) gives a value of
16.2 kcal/mol, which is a decrease of over 40 kcal/mol compared to a transfer to a hydrocarbon
environment. When the actual shape of the cavity and the fact that the bulk water is not infinitely
far away is taken into account the authors arrive at a value of 6.3 kcal/mol. The fact that two K+
ions are present in the selectivity filter increases this value with another 10 kcal/mol. The transfer
energy drops to –8.5 kcal/mol when all the atomic charges of the protein and the presence of the
selectivity filter ions are turned on. The authors stated that dielectric shielding by water molecules
in the cavity minimizes the electrostatic influence of the helix dipole moments, 8 Å away from the
cavity center. They add, however, that the low dielectric membrane environment contributes
significantly to the amplification of cation stabilization. They calculated that the helices are
responsible for eighty percent of the stabilization, leaving the other twenty percent to a Thr carbonyl
group.[19]
Using the model proposed by Doyle et al., Zhou et al. sought to answer two additional
questions regarding the Ksca K+ channel. The first is how the K+ channel mediates the transfer of a
K+ ion from its hydrated state in solution to its dehydrated state in the selectivity filter. This is relevant to all mechanisms of selective ion transport. Because dehydration in the wrong environment is
energetically costly, the authors went to look for a set of mechanisms designed to handle hydrated
K+ ions and also mediate their dehydration. The second question addressed in this study is related
to the cellular environment in which K+ channels operate: inside the cell the K+ concentration is
greater than 100 mM, whereas on the outside the K+ concentration is usually less than 5 mM. By
solving structures at both high and low K+ concentrations, the authors were able to conclude that
two conformations of the channel occur[23].
To return to the first question; The hydration of K+ in the cavity and the conformation of the
carbonyl atoms arranged to replace the hydration appear to be precisely regulated. The cavity in
KscA and most other K+ channels is lined mainly by hydrophobic amino acids from the inner helix
and hence no strong hydrogen-bonding donor or acceptor groups are available for the water molecules surrounding the ion. Consequently, water in the cavity is available to interact strongly with the
K+ ion. Eight water molecules surround the K+ ion in a square antiprism conformation. This orientation will later be mimicked in the selectivity filter by carbonyl groups. Regarding the question of
how this precise ordering is conducted, the authors conclude that inspection of the cavity wall
shows that the order is probably imposed by a sum of very weak, indirect hydrogen-bonding interactions mediated by certain chemical groups, and perhaps the carbonyl oxygen atoms from the pore
and inner helices. The cavity achieves a very high effective K+ concentration (2 M) at the center of
the membrane, with the K+ ion positioned on the pore axis, ready to enter the selectivity filter.[23]
Dutzler et al. Determined the structure of two CIC chloride channels. The resolution of these
structures, originating from Salmonella enterica serovar typhimurium and Escherichia coli, is 3.0
and 3.5 Å respectively. While the biological role of this class of chloride channels remains
unknown in many cases, they are widespread in both eukaryotes and prokaryotes. In vertebrates
they account for stabilizing the membrane potential in skeletal muscle cells and regulation of
excitability. Because of the sequence conservation that is observed among different organisms it is
highly plausible that the mechanisms of ion submission are equal. Both channels are double-barrels,
consisting of two dimers bound in an antiparallel fashion. This antiparallel arrangement differs from
the architecture of K+ channels, but is also found in aquaporins. This architecture enables both ion
stabilization by N-termini at the inside of the channel and location of C-termini in the aqueous
environment outside of the membrane, leading to both ion stabilization and avoidance of the
energetically costly burial of the C-terminus in an hydrophobic environment. Although at present
only dimers appear to be functional channels each unit has its own pore and selectivity filter.[5]
A comparison of amino acid
sequences of both units show that
the units are slightly correlated
especially with respect to glycines.
Transmembrane helices of both units
wrap around a common center,
thereby bringing the ends of
different helices together. The Ntermini of two helices appear to be
directed towards a Cl- binding spot.
Other atoms thought to be involved
in ion coordination are Ile and Phe
main-chain amide nitrogen atoms Figure 5: The anti-parallel orientation allows like helix ends to point at the
and sidechain oxygen atoms from selectivity filter from opposite directions [5]
Ser and Tyr. The authors suggest that
coordination by partial charges permits rapid diffusion rates, whereas using full charges would
result in a deep energy well. A second ion binding spot is located at the extracellular side of the
channel. This spot is occupied by a carboxylate ion and the authors suggest that this spot is
important for the strong coupling between gating and ion conduction that is a general characteristic
of CIC Cl- channels. Chloride ions compete for this second binding site. Upon binding, certain
conformational changes take place in the selectivity filter so that the sidechain of a conserved Glu
blocking the pore swings out of the way. The chloride bound at the inner site is thought to play an
important role in the ion selectivity of the channel, thereby giving rise to properties such as voltagedependent gating of some ClC Cl- channels.[5]
Faraldo-Gomez and Roux examined the contribution to the electrostatic binding free energy
of local and non-local interactions of EcClC, a ClC-type chloride channel homologue from Escherichia coli and made a comparison with the stabilization in K+ channels and sulphate and phosphatebinding proteins (SBP and PBP). While the helix dipole is often mentioned as an important contributant to energetic stabilization of ion species, as we have already seen, various theoretical studies,
suggest otherwise, concluding that the stabilization is due to local interactions. Hence the comparrison made by the authors. The authors conclude that the contribution of helix macrodipoles to chloride binding in EcClC is only marginal and is analogous to the binding mechanism of the sulphate
and phosphate-binding proteins. The transmembrane potassium channel KcsA in contrast seems to
profit from helical stabilization in a significant way. The following reasons may account for this.
Firstly, the low dielectric environment provided by the membrane for example helps to magnify the
stabilizing effect, a fact that was also recognized by Roux and MacKinnon[19]. Calculations based on
a situation where the membrane is absent show a decrease in stabilization by 50%. The distant ionbackbone interactions with the cavity ion in K+-channels, with the helix being 8 Å from the ion,
come mainly from the pore helices and are prominent simply because more proximal interactions do
not occur. EcClC is topologically more similar to SBP and other soluble ion-binding proteins, whereas its environment is analogous to that of KcsA. Binding of anions within PBP, SPB and ClC
channels is largely due to favorable electrostatic interactions with the backbone of the protein. These interactions must at least balance the energetic cost of desolvation. In both PBP and SPB the binding site is located between the N-termini of three α-helices. Calculations show that only the first
(80%) and second (95% for the first two turns together) turn contribute to the stabilization. The different relative importance of helical macrodipoles for the energetic stabilization of ions in K+-channels and the SBP and PBP proteins can therefore be understood on the basis of the distinct architecture and environment of these proteins.[6]
Chatelain et al. obtained results contradicting the results of these calculations, and hence the
mechanistic models of Doyle and Dutzler[4, 5]. They conclude that helix dipoles have a negligible
role in electrostatic stabilization and channel function. They introduced several mutations in yeast
potassium channels. The C-termini of helices were modified by introducing positively charged
residues at a conserved position, thereby canceling out the electrostatic effect of the helix. Diffusion
rates vary for each mutation but generally mutated channels function well, indicating that potassium
ions are largely blind to electrostatic perturbations at the C-terminal end of the pore helix. They also
question the conclusion of Roux and MacKinnon[19], stating that electrostatic effects over a distance
of 8 Å even exceed the distance covered by ion-dipole interactions. Chatelain et al. suggest that the
stabilizing effects of the helix, if any, arises from unpaired hydrogen bonding groups near the termini that act over very short distances.[3]
Aquaporins
The usage of helix dipoles in selectivity filters is also proposed by Murata et al. Using electron
crystallographic data, he and his coworkers determined the structure of the aquaporin AQP1 at a 3.8
Å resolution. This is a water-selective membrane pore found in red blood cells and renal proximal
tubules. Other members of the aquaporin family are found throughout nature where they are involved in numerous physiological processes. The complete channel consists of a tetramer of identical
subunits, each being 269 amino acids in length and forming three membrane-spanning helices.
Besides its exceptional water permeability of over 3 x 109 molecules per second, its ability to
prevent any protons from crossing is intruiging. It is known that water can readily cross cell
membrane as a continuous unbroken column of molecules within an open pore. This hydrogenbonded chain of water molecules normally conducts protons with great efficiency. Based on the
structure the authors conclude that two pore helices, HB and HE, play a vital role in this mechanism
for distinguishing. Because of the resolution no water molecules (2.8 Å) could be distinguished, so
this conclusion remains hypothetical. The shape of the AQP1 pore is the opposite of the pore shape
formed by the potassium channel, having a constriction in the centre of the membrane and wide
openings at the membrane surfaces. This constriction results in a high dielectric barrier that repels
ions, while it allows for penetration by neutral solutes. Both pore helices have an adjacent Asn-ProAla motif that is highly conserved. Prolines of these motifs interact to ensure the orientation of the
pore helices and to maintain the diameter of the pore at 3 Å. Due to the positive electrostatic field
generated by the dipole moments of the pore helices, the oxygen atom of a passing water molecule
orients to the side of this motif, whereas the hydrogen atoms are directed towards hydrophobic
residues lacking the ability to form hydrogen bonds. This comes at an energetic cost of 3 kcal/mol,
since only one hydrogen bond is lost compared to a bulk water environment. Because of this
hydrogen bond isolation, the water column is disrupted. Thus, protons no longer can pass the
channel. This mechanism is brightly illustrated in Figure 6[14].
Fu et al. state that at present, ten aquaporins of
the human family are known, all of which are
permeable to water. AQP3, AQP7, and AQP9 are
also permeable to glycerol. Glycerol permeabilty
results from a mchanism quite similar to that of
AQP1, albeit that carbon atoms of glycerol are
aligned to a hydrophobic wall whereas the oxygen
atom of water is coordinated by a helix dipole. In
eukaryotes many of the family members are
regulated by phosphorylation, pH, osmolarity, or
binding of other proteins or ligands. AQP6
conducts cations at pH values below 5,5.[7]
Quantifying the dipole moment
In 1988, Šali, Bycroft and Fersht quantified the
electrostatic stabilization energy of various forms
of barnase, a small ribonuclease found in Bacillus
amyloliquefaciens, by NMR-titration. The enzyme
contains a histidine at the C-terminal end of a
twelve amino acid long α-helix. The authors
reported that the pKa of the wild-type enzyme is
Figure 6: Representation of AQP1 and its mechanism to 7.9, whereas titration of the urea-denatured
disrupt the water column by forming alternative hydrogen enzyme results in a pK of only 6.3. At a neutral
a
bonds with the channel.[14]
pH, the stabilization is therefore 2.1 kcal/mol. The
authors also conducted an experiment adding 1M KCl, a solution known to counter the charges of
individual protein sidechains, to rule out these effects in the stabilization. No sidechain involvement
was found, as the structure already predicted.[20]
Another effort to calculate the strength of the electrostatic effect of the α-helix dipole was
carried out by Lockhart and Kim. They introduced a probe, 4-(methylamino)benzoic acid (MABA),
at the amino terminus of a protein whose absortion bands could be measured. Because the difference between the ground state and the excited state of MABA is known, the electric field could be determined by measuring the shift in the absorption band using Stark effect spectroscopy.[11]
The authors synthesized a range of peptides that differed in length, amino acid composition,
resulting in folding and non-folding proteins and the occurrence of Arg. 2D-NMR and circular dichroism were applied to check that the proteins that should be were properly helical. The authors
checked the position of the probe using NMR. It was found that in the presence of a helical structure, the absorption band of the MABA probe is shifted by -5 nm. This band shift corresponds to a
change in the transition energy of 1.6 kcal/mol. Similar values were found with other proteins, indicating that the observed field is primarily produced by backbone dipoles, not sidechain charges, and
that the magnitude of the field is independent of the helix length between 21 and 41 residues.
Although diminished by the environment, the field measured at the boundary between the peptide
and water is still an order of magnitude stronger than expected based on the dielectric properties of
bulk water, which has a dielectric constant of 88 at 0°C. A dielectric constant between 2 and 4 is
usually used for the interiors of proteins. A larger effective dielectric constant, between 30 and 100,
is considered appropriate for interactions involving unpaired charges, especially if the charges are
solvent-accessible. The authors concluded that a value of eight should be appropriate for estimating
the strength of short-range dipole-dipole interactions that occur near protein surfaces, such as those
involved in ligand binding and enzymatic reactions.[11]
In his review ‘Roles of electrostatic interactions in proteins’ (1996), Nakamura mentions a
number of articles where the stabilizing influence of the helix dipole in short peptides is
demonstrated. First of all there is the C-peptide, a thirteen residue fragment of ribonuclease A. It
was found that Glu 2 and His 12, being negatively and positively charged respectively, are
responsible for the stability of the peptide. Experiments were also conducted on the slightly larger
fragment called the S-peptide. Various charges were introduced at the N-terminus, and the stability
increased as the charge changed from +2 to -1. All experiments mentioned in the review agreed on
the fact that helices do not necessarily have to be long to have a significant charge effect, even when
the dielectric shielding effect was taken into account. The author concluded, based on the results of
Aqvist and coworkers[1], that only the first one or two turns of an α-helix are likely to participate,
and bring along a stabilization of at most about 2 kcal/mol, which is less then the estimate made by
Hol.[8, 15]
Following the discovery that the electrostatic field is drastically lowered by the field
generated by the solvent, Sengupta et al. tried to calculate and quantify the net effective dipole
moment using atomic-detail helix models and Poisson-Boltzmann continuum electrostatics calculations. Helices of varied lengths were used in different environments (vacuum, aqueous solution, lipid bilayers and protein interiors). The authors concluded that although the helix dipole is quite
strong in vacuum, its strength is drastically reduced in aqueous solution due to the reaction field that
is generated by the solvent. Further, while the net dipole moment increases with helix length in vacuum, the opposite is true for transmembrane helices, where an increasing length brings the termini
closer to the shielding solvent. The authors state that the dipole moment of a helix can be used to
determine electrostatic interactions at distances that are large compared to the dipole length and thus
is likely inadequate for describing close range effects such as phosphate binding and antiparallel helix-motif stabilization. The authors postulate three interwoven rules-of thumb that follow from their
results. The first one is that the dipole strength is determined by the position of the helix termini
compared to the aqueous phase, whereas the total solvent-exposed area is less important. The second rule states that if both helix termini are solvent exposed, the net dipole moment will be small,
almost equal to the situation where the helix is fully solvated. The third rule says that in cases where
one helix terminus is solvent exposed and the other buried, asymmetric reaction field shielding can
lead to a relatively high net dipole moment.[21]
Lorieau and coworkers also reported an unexpected increase of pKa value for a highly
conserved N-terminal glycine in the the influenza coat protein hemagglutinin HA2, which is known
to mediate the fusion of viral and host-cell endosomal membranes. Two helices form a tight hairpin,
stabilized by four interhelical hydrogen bonds. The authors argue that the reported pKa of 8.8 of the
glycine is also due to interactions between the glycine and the dipole of the second helix. 3D NMR
was applied, because protonations lead to a change in chemical shift. While the protonation state of
the glycine was still in debate, the authors proposed the glycines amino group is in fact protonated.
Normally, the pKa value of an N-terminal amino group of an alpha-helix in aqueous solution is
depressed by about 0.5 units by the positive potential imposed by the helix dipole moment.
Embedding of the peptide in the hydrophobic environment of the viral membranes would be
expected to decrease its pKa value even further. However, a potential α-NH3+ interaction with one
of the carboxyl groups of the peptide or with the phosphate of the lipid headgroup was proposed as
a possible reason for the elevated pKa of this glycine.[12]
Allosteric mechanisms
In 2009, Preininger et al. demonstrated that electrostatics are also used to initiate nucleotide release
in the mechanism of nucleotide exchange in G-proteins. Heterotrimeric G-proteins transmit signals
from activated G protein-coupled receptors to downstream effectors through a guanine nucleotide
signaling cycle. After the receptor binds its cognate G protein, the signal is transmitted from the
receptor-binding site to the nucleotide binding pocket of the Gα-subunit. Numerous studies already
suggested that the C-terminal α5 helix of Gα-subunits participates in Gα-receptor binding and that
this binding process induced a rotational change in the same helix. Electron paramagnetic
resonance (EPR) spectroscopy data indicated that the α5 helix rotation could be communicated to
the nucleotide binding site both via global structural effects and through specific electrostatic effects
directed at the base of the α5 helix, which moves closer to the bound guanine nucleotide upon rototranslation of the α5 helix. Using this knowledge and the protein structure of the Gα-subunit, the
authors introduced two cysteines in the protein to lock the protein into its receptor-associated
conformation via a disulfide bond. The 2.9 Å crystal structure of the protein in complex with GDPAlF4 proved the success of this disulfide bond. Because the roto-translation of the α5 helix would
also be expected to move the positive dipole of the α5 helix toward the guanine ring of the bound
nucleotide the authors constructed a second complex where a positive charge was introduced at the
N-terminus, next to the TCAT-motif that is known to be involved in nucleotide binding. Since the
negatively charged guanine ring may be attracted by the positively charged helix dipole, this
construct was thought to lead to an increase in nucleotide exchange. Besides this front door exit for
guanine, a back door mechanism may also exist. However, the authors cannot rule out one
mechanism based on these experiments and additionally no helix dipole interactions are thought to
be involved in the back door mechanism.[17]
An example of a mechanism to switch between conformations where a helix dipole is
involved is described by Neuwald. He analyzed both amino acid sequences and structures of several
Ras-like GTPases. The on- and off-states of these GTPases is communicated through
conformational changes in the switch I and II regions. Neuwald describes an additional regulatory
structural element found in a subgroup of GTPases. At the end of the switch II helix an aromatic
pocket is found with a positively charged residue inserted into it, facing the N-terminal side of the
helix. This helix is pointed away from the GTPase core. Five residues that form the aromatic pocket
are found to be highly conserved in these GTPases. The aromatic pocket is found in both the onand off-state. When this aromatic
pocket is disrupted, the direction of the
helix changes and comes along with a
restructuring of the entire switch II Nterminal domain. This domain is
known to sense the γ-phosphate of
GTP and to harbor three previously
noted Ras-like residues involved in
GTP hydrolysis and nucleotide
exchange. An association between the
charge-dipole pocket and switch II
restructuring is suggested by
comparisons between typical
Figure 7: The charge dipole pocket of GDP-bound Rab11a (PDB ID 1oiv,
monomeric forms of Rab family
indicates the characteristic atomic interactions of
GTPases and an unusual homodimeric resolution 1.98 Å).The inset
the charge dipole pocket.[16]
form, Rab27, where the inter-switch
region that connects switch I to switch II is exchanged between subunits. The switch II region forms
a long α-helix that is directed away from the structural core of one subunit and toward the structural
core of the other subunit. Other GTPases lack both the charge-dipole pocket configuration and the
outward directed switch II helix. In addition, for GDP-bound Rab11a and Rab23, minor deviations
from the charge-dipole pocket configuration are associated with shortening or lengthening of the
outward-directed switch II helix, suggesting that this helix can extend and retract. Together, this
indicates that the charge-dipole pocket configuration is closely associated, not with the on- or offstate, but rather with formation of the outward-oriented helix which, in turn, is associated with
restructuring of the switch II N-terminal region and, as a result, with repositioning of co-conserved
residues implicated in GTP hydrolysis and nucleotide exchange. Figure 7 shows the charge-dipole
pocket and their interactions.[16]
Somewhat similar to this protein is the bacterial transcription regulator YvoA, part of the
GntR/HutC family which is investigated by Resch et al. At their N-terminus they contain a winged
helix–turn–helix domain which is responsible for DNA binding. Both domains of the dimer are in
close proximity in the DNA-bound conformation, binding in successive major grooves. The Cterminal domain of one unit of this homodimeric protein contains a chorismate lyase-type UTRA
domain. Whereas this domain functions as an active site in other species, here it constitutes a
binding pocket for various effector molecules.[18]
This domain encompasses an allosteric mechanism that alters the affinity of the DNAbinding domains for DNA upon binding of an effector. Resch et al. introduced disulfide bonds to
lock the regulator either in its DNA-bound or the effector-bound conformation in order to
investigate the mechanism that triggers the transition between these conformations and its
consequences for DNA binding affinity. The authors determined the structure with the transcription
regulator bound to an effector, GlcNAc-6-P. They propose that the negatively charged phosphate
group both stabilizes the helix dipole of one helix and induces the formation of another helix whose
dipole also points toward the
phosphate group. The new helix
again induces a helix formation
which generates internal
symmetry in the effector binding
domain and is proposed to be a
key step in the propagation of
the allosteric signal. The DNAbinding domains are then
reoriented, rotating 122 degrees, Figure 8: The two conformations of YvoA[18]
and hence unable to bind DNA. This mechanism, which the authors describe as a 'jumping jack'-like
motion, is summarized in Figure 8.[18]
Other helix types
Another type of helix occurs in nature is the polyproline II helix, which is involved in biological
processes such as signal transduction, transcription, immune response, and cell motility. Strands of
collagen with the [Pro-Hyp-Gly]n repeat unit also adopt a PPII-like conformation. Kuemin et al.
examinated the the influence of different terminal groups on the stability of this type of helix. Peptides with a PPII structure can switch to a PPI strucure, and the ease with which this occurred was
used to measure differences in stability. An ideal PPII helix has dihedral angles φ and ψ of -75° and
+145°, respectively, and all amide bonds are in trans conformations (ω = 180°). This results in a
left-handed helix with every third residue stacked on top of each other in a lateral distance of 9.4 Å.
Within this helix, all amide bonds of the peptide backbone are nearly perpendicular to the helix
axis. The PPI helix is right-handed, and all amide bonds are in cis conformations with φ and ψ angles of -75° and +160°, respectively. These dihedral angles result in a more compact structure (helical pitch of 5.6 Å per turn, 3.3 proline residues per turn) with the amide bonds oriented nearly parallel to the helix axis. In contrast to the α-helix, neither the PPII nor the PPI helix is stabilized by intramolecular hydrogen bonds.[10]
Four oligopeptides were used, similar in length but either carrying no terminal charges (at
pH 7.0), a C-terminal negative charge, an N-terminal positive charge or both charges at the same
time (AcN-[Pro]12- CONH2 (1), HN-[Pro]12-CONH2 (2), AcN-[Pro]12-CO2H (3), and HN[Pro]12-CO2H (4)). The PPII helix is the predominant conformation of oligoprolines in water, whereas the PPI helix is favoured in solvents such as n-PrOH. The amount of n-PrOH needed to switch
the PPII helix to the PPI helix therefore reflects the ease of the conformational change between the
PPII and the PPI form. The oligopeptides were studied in different mixtures of aqueous phosphate
buffer (10 mM, pH 7.2) and n-PrOH by CD spectroscopy. The results revealed that capped termini
stabilize the PPII helix, whereas charges have a destabilizing effect. This results from the fact that
within the PPI helix, the individual dipoles are oriented almost linearly to the helix axis, and also
being more compact. Moreover, the negative charge at the C-terminus has a more pronounced destabilizing effect as compared to a positive charge at the N-terminus. (95 vol % n-PrOH to switch
whereas 85 vol % n-PrOH suffices for the C-terminal charge). The authors relate this difference to
destabilizing effect by a repulsive interaction of the carboxylate with the oxygen of the neighbouring amide.[10]
Supramolecular interactions
Another example of interstrand stabilization is found in invertebrate collagen. Collagen is a
supramolecular structure composed of three helices of individual strands. Shoulders et al. state that
the amino acid composition of common collagen is composed of Gly-Xaa-Yaa repeats. Stable
collagen triple helices form when (2S)-proline or Pro derivatives that prefer the Cγ-endo ring pucker
are in the Xaa position and Pro derivatives that prefer the Cγ-exo ring pucker are in the Yaa position.
Shoulders and Raines reported a form of collagen found in invertebrates that has a Cγ-exo–puckered
Pro derivative ((2S,4R)-4-hydroxyproline, or Hyp), in the Xaa position. The triple helix is found to
be hyperstable. After synthesizing analogous proteins lacking the possibility of obtaining additional
stabilization through hydrophobic effects and increased hydrogen bonding[22]. A computational
analysis by Improta, Berisio, and Vitagliano suggested that interstrand dipole–dipole interactions
could more than compensate for the energetic penalty of triple-helix distortion caused by the
incorporation of a Pro derivative with a Cγ-exo pucker in the Xaa position[9]. This proved to be the
case, after synthesizing analogues where the presence of a favorable dipole–dipole interaction was
the constant. The authors suggest that analogous strategies could be applicable in other contexts
such as coiled-coils.[22]
The supramolecular of collagen already made clear that the electrostatic effects of the helix
dipole are not limited to intramolecular cases. Another case to illustrate this is the binding
mechanism of centrin. Centrins are acidic Ca2+-binding proteins that are well conserved in the
eukaryotic realm. Three isoforms of human centrin exist, with all isoforms containing two EF-hand
motifs and target binding sites. Martinez-Sanz et al. examined the effects of a reversal of the
traditional target sequence, and thus the reverse of the helix dipole, W1L4L8 on the dynamics and
affinity of a single centrin isoform, HsCen2. A specific target sequence was chosen because an
already determined protein structure featured this same sequence, albeit in the normal orientation.
HsCen2 has the ability to bind any of the ∼25 repeats of this sequence, found in human Sfi1, with
more or less affinity. As only the C-terminus was involved in the binding of the Sfi1 repeat, the
structure was determined for the C-terminal domain only. The Trp in the target sequence is normally
embedded in a hydrophobic cavity of HsCen2. With the reversed target sequence, this still proved to
be the case, although the χ1 dihedral torsion angle switched from −80° to −160° . Another
consequence was a ∼2 Å translation of the Sfi1 sequence towards a highly conserved glutamate
sidechain, which is also conserved in calmodulin and troponin molecules for probably the same
reasons. This resulted in a the lower enthalpy ΔΔH ≈ –7 kcal/mol and a lower free binding energy
ΔΔG ≈ –2 kcal/mol compared to the normal orientation of Sfi1. This was measured using
isothermal titration calorimetry. It is an aggregate effect, due to the a slight loss of hydrophobic
interactions with Leu 8 and a inversion of the helix dipole that leads to a positive charge pointing at
the conserved glutamate residue. The first of these probably exerts a negative effect on the calcium
affinity of loop III of the centrin.[13]
Conclusion
Since the discovery of the helix dipole and its relevance to various biological structures and
mechanisms, the subject has gained a serious foothold in the scientific literature. The exact amount
of influence in reducing energetical costs, however, is still under debate. A number of researchers
conclude, using both theoretical approaches and calculations and experiments, that only the termini
are involved in this stabilization which according to them mainly originates from hydrogen
bonding. Nevertheless, its overall biological relevance is widely acknowledged
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